Original Contribution
Kitasato Med J 2015; 45: 9-16　Effects of hydrogen water on paraquat-induced
pulmonary fibrosis in mice
Chie Sato,1 Yoshito Kamijo,1 Kuniko Yoshimura,1 Taito Inagaki,1 Tatsuhiro Yamaya,1
Sadataka Asakuma,1 Masataka Majima,2 Yasushi Asari1
1
2
Department of Emergency and Critical Care Medicine, Kitasato University School of Medicine
Department of Pharmacology, Kitasato University School of Medicine
Objective: Chronic paraquat (PQ) intoxication causes pulmonary fibrosis. PQ-induced pulmonary
injury is caused by reactive oxygen species (ROS) that are produced during the reduction-oxidation
(redox) cycle. In addition, the hydrogen has antioxidative effects. The present study aimed to
determine whether or not the intake of hydrogen water (HW) prevents pulmonary injury caused by
chronic PQ intoxication.
Methods: PQ was administered to 10-week-old male C57BL/6 mice, and the mice were randomly
assigned to either the HW group (PQ + HW group) or the tap water group (PQ group). After 3 weeks,
the mice were assessed for pneumodynamic and histologic changes.
Results: Pneumodynamic analysis revealed that elastance (E) (P = 0.010) and hysteresivity (eta) (P =
0.048) were significantly lower in the PQ + HW group than that in the PQ group. Although no
significant difference was observed in compliance, it tended to be higher in the PQ + HW group.
Histologic findings showed that inflammatory cell infiltration and fibrosis were comparatively lower
in the PQ + HW group.
Conclusion: The results of the present study suggest that HW may prevent the development of PQinduced pulmonary injury.
Key words: paraquat-induced pulmonary fibrosis, hydrogen water
Ohsawa et al. have previously reported that the
antioxidative effects of HW are produced through the
selective reduction of strong oxidizing ROS such as
hydroxyl radicals and peroxynitrite.8 The methods of
administration of hydrogen (H2) include inhalation of H2
gas, drinking HW, and intraperitoneal or intravenous
administration of saline solution with dissolved H2. H2
has already been shown to be effective in animal
experiments involving cisplatin-related renal damage,9
atherosclerosis,10 and metabolic syndrome.11 Clinical
studies on the amelioration of Parkinson's disease are
currently being conducted.12
Various models of pulmonary injury have been
prevented by H2.13-15 Therefore, it is expected that H2 is
effective for PQ-induced pulmonary injury.
We administered PQ to mice and comparatively
examined those that were treated with HW and those that
received tap water. We then performed pneumodynamic
and histologic assessments of the effects of oral
Introduction
araquat (PQ) (1,1'-dimethyl-4,4'-bipyridinium
dichloride) has been used as an herbicide for many
years in Asian countries, including Japan. However, due
to a large number of fatal accidents, the concentration of
PQ in various products has been lowered, and it is now
mandatory to present a photograph ID to purchase PQ
herbicides. Nevertheless, patients are still sometimes
admitted to the hospital for attempted suicide by PQ
poisoning. The mortality rate for PQ poisoning is as
high as 60%, and patients who survive the acute poisoning
often die later due to respiratory failure.
When PQ enters the cells, it undergoes repeated
reduction-oxidation (redox) cycling, which produces a
variety of reactive oxygen species (ROS) leading to
irreversible fibrosis of pneumocytes.1-7 On the other hand,
hydrogen water (HW), a colorless, tasteless, and odorless
gas, has been widely reported for its antioxidative effects.
P
Received 26 September 2014, accepted 8 October 2014
Correspondence to: Chie Sato, Department of Emergency and Critical Care Medicine, Kitasato University School of Medicine
1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan
E-mail: [email protected]
9
Sato, et al.
hypodermic needle was inserted into the 4-mm incision,
and flexiVent (SCIREQ Scientific Respiratory
Equipment, Montreal, Canada), a high-performance
respiratory function analyzing computer system, was
loaded. An incision was made on the diaphragm to stop
spontaneous respiration and was ventilated at a rate of
150 beats/minute and at a volume of 10 ml/kg. To prevent
lung collapse, PEEP (positive end-expiratory pressure)
at 2−3 cm H2O was applied.
First, for total lung capacity (TLC), the lungs were
inflated to 30 cm H2O for 3 seconds to stabilize the lungs.
Snapshot measurements were then taken to calculate
resistance (R), compliance (C), and elastance (E) of the
respiratory system as a single compartment model, which
includes the trachea, lungs, and thoracic wall. The pistons
of the ventilator were spun multiple times at a fixed pace,
and the resulting volume changes in quasi-sinusoidal and
airway pressures measured at this time were used to
calculate R, C, and E.
Forced oscillation technique (FOT) measured
respiration that occurred at random frequencies (0.25−
20 Hz), and Newtonian resistance (Rn), tissue damping
(G), tissue elastance (H), and eta (hysteresivity) were
measured for each frequency. A comparison of these
parameters in the central and peripheral airways was then
performed.
TLC, Snap Shot (SCIREQ Scientific Respiratory
Equipment), and FOT were continuously measured and
conducted in triplicate. The data were analyzed using
the flexiVent software.
administration of HW on PQ-induced pulmonary injury.
Materials and Methods
Animals
Ten-week-old male C57BL/6 mice (CLEA) were used.
The mice were placed in a room with the appropriate
humidity and temperature and acclimated for 1 week
using a 12-hour light/12-hour dark cycle. The mice were
randomly assigned to two groups; one group received
PQ and HW in their drinking water (PQ + HW group, n
= 12) and the other group received PQ and tap water (PQ
group, n = 9). All animal experiments were performed in
accordance with the guidelines for animal experiments
of the Kitasato University School of Medicine (2014048).
PQ ingestion
An anesthesia system (SN-487-OT air + O2; Shinano,
Tokyo) designed for laboratory animals was used for the
administration of isoflurane (Forane; Abott Japan, Tokyo)
via inhalation. An anterior cervical T-shaped incision
was then created to fix the trachea onto the surface of the
body. An intratracheal aerosolizer (MicroSprayer, IA1C-M; PennCentury, Pennsylvania, USA) was inserted
into the oral cavity and directed into the trachea. Upon a
visual confirmation of the presence of the aerosolizer in
the trachea, a 20-μl dose of a 0.25 mg/ml PQ solution
(Tokyo Kasei Kogyo, Tokyo) was intratracheally
administered. After decannulation, the wound was closed
using nylon sutures.
Lung histopathology
After performing flexiVent measurements, the mice were
euthanized using an overdose of pentobarbital, an
anesthetic agent. For histological assessment, the lungs
were isolated, fixed with 4% paraformaldehyde, and then
embedded in paraffin. Lung sections of 3-μm thickness
were then prepared and subjected to hematoxylin and
eosin (H&E) and Heidenhain's Azan staining.
HW
HW (Blue Mercury, Tokyo) containing supersaturated
molecular H2 at a pressure of 0.4 MPa was used. HW
was transferred from an aluminum bag to a closed glass
vessel connected to a double ball-bearing water pipe and
silicon cap, allowing the mice to drink water ad libitum.
The bottles were changed once a day and residual H2 gas
was monitored using a dissolved H2 analyzer (HMD-X1;
Hikari Bellcom, Kanagawa). Ohsawa et al. used a similar
method for the administration of HW, and an H 2
concentration of ≥0.4 mM was maintained after the first
day using an H2 electrode.10 Tap water was provided by
an automatic water feeder, allowing the mice to drink
water ad libitum.
Statistical analysis
All data were expressed as the mean ± standard error of
the mean. Both groups were compared using the unpaired
student's t-test. A P-value of <0.05 was considered to
indicate statistical significance.
Results
Measurement of lung function
Three weeks after intratracheal administration of PQ,
pentobarbital was intraperitoneally administered and a
tracheal incision was made under anesthesia. An 18 G
Lung function
We comparatively examined the PQ + HW and PQ groups
to ascertain whether or not HW could reduce PQ-induced
10
Hydrogen water for paraquat-induced pulmonary fibrosis in mice
Figure 1. Snap Shot (SCIREQ Scientific Respiratory Equipment)
Lung function 3 weeks after the administration of PQ by Snap Shot showed: A. resistance (R),
B. elastance (E), and C. compliance (C). *P = 0.010
Figure 2. FOT
Lung function 3 weeks after the administration of PQ by FOT showed A. Newtonian resistance
(Rn), B. tissue damping (G), C. tissue elastance (H), and D. hysteresivity (eta). *P = 0.048
11
Sato, et al.
Figure 3. Histopathological analyses
Pulmonary histologic findings 3 weeks after PQ
administration. A. (PQ group) and B. (PQ + HW group)
showing H&E staining. Compared with the PQ group,
the PQ + HW group showed lower levels of edema,
inflammatory cell infiltration, and alveolar cell
hypertrophy (Original magnification ×200). A-1. The
arrows indicate the inflammatory cell infiltration
(Original magnification ×400). C. Slight
emphysematous change (Original magnification ×
200). D. (PQ group) and E (PQ + HW group) showing
Azan staining. Compared with the PQ group, the PQ +
HW group showed milder fibrosis (Original
magnification ×200). D-1. Arrows indicate fibrosis
(Original magnification ×400).
12
Hydrogen water for paraquat-induced pulmonary fibrosis in mice
pulmonary injury. The Snap Shot results are shown in
Figure 1. No significant difference in R was observed
between the PQ + HW and PQ groups (0.507 ± 0.03 vs.
0.577 ± 0.054, respectively; P = 0.249) (Figure 1A). E
was significantly lower in the PQ + HW group than that
in the PQ group (19.506 ± 0.491 vs. 21.569 ± 0.56,
respectively; P = 0.010) (Figure 1B). No significant
difference was detected for C between the PQ + HW and
PQ groups, although C was higher in the PQ + HW
group compared with that of the PQ group (0.051 ±
0.001 vs. 0.047 ± 0.001, respectively; P = 0.0628) (Figure
1C).
The results of FOT are shown in Figure 2. No
significant difference in Rn was observed (0.297 ± 0.025
vs. 0.307 ± 0.033, respectively; P = 0.806) (Figure 2A).
No significant significance was observed for G and H
(G, 2.565 ± 0.202 vs. 2.939 ± 0.187, respectively, P =
0.204; H, 20.345 ± 0.927 vs. 21.764 ± 1.073,
respectively, P = 0.326) (Figures 2B and C), although eta
was significantly lower in the PQ + HW group than that
in the PQ group (0.120 ± 0.009 vs. 0.146 ± 0.008,
respectively, P = 0.048) (Figure 2D).
effects of vitamin C (ascorbic acid), vitamin E (αtocopherol), melatonin, glutathione (GSH), Nacetylcysteine, and iron chelators have all been studied.
SOD, vitamin C, vitamin E, and GSH were unable to
cross the cell membrane barrier, and their clearance
appeared to be limited or not at all occurring.7
The effects of cyclophosphamide and glucocorticoids
were negated in the largest randomized controlled study
consisting of 298 patients.17 Earlier in vitro and in vivo
studies have shown that melatonin, N-acetylcysteine, and
iron chelators prevent PQ poisoning; however, they have
not yet been therapeutically used in humans. 5,18-20
Therefore, although an antidote for PQ has been shown
to be effective in animal experiments and studies, no
antidote for humans has been identified.
As PQ undergoes the redox cycle, it produces ROS,
which has been previously shown to be correlated with
ischemic reperfusion injury, Parkinson's disease,
arteriosclerosis, metabolic syndrome, and aging. ROS
possess hydroxyl radicals (・OH) and peroxynitrite
(ONOO-), which have strong oxidative powers, as well
as interact and damage DNA, proteins, and lipids.
However, superoxides (・O2-) and hydrogen peroxide
(H 2O 2 ) have signal transduction functions at low
concentrations,21-23 making them extremely important.
Antioxidants such as vitamin C, which has a strong
reducing power, can eliminate all ROS, which disrupts
the signal transduction pathways in the body.
Furthermore, vitamin C produces substances with strong
oxidative power after redox reactions, resulting in DNA
damage.24,25
Because the reducing power of molecular H2 is weak,
it only reduces ROS with a strong oxidizability such as
hydroxyl radicals (・OH) and peroxynitrite (ONOO-).8
Furthermore, because the amount of H2 is small, it can
easily cross the cell membrane and reach the nucleus and
mitochondria; thus, protecting the cell from oxidation.
Previous studies have shown that H2 is safe. H2 gas
has been used for the prevention of decompression illness
in divers. 26-28 Furthermore, there were various
administration methods for H2 such as drinking HW water,
the inhalation of H 2 gas, and the intraperitoneal or
intravenous administration of normal saline solution
containing H 2, which have all been shown to have
antioxidative efficacy.29
The administration of HW to animal models has been
shown to be effective for the treatment of Parkinson's
disease,30,31 atherosclerosis,10 metabolic syndrome,11 and
cisplatin induced nephrotoxicity.9,32 The administration
of hydrogen gas has been shown to improve prognoses
for cerebral infarction,8 myocardial infarction,33 and
Histopathological analyses
Figure 3 shows the pulmonary histologic findings for the
PQ + HW and PQ groups. In the PQ group, edema,
inflammatory cell infiltration, and alveolar wall
hypertrophy were observed (Figure 3A, A-1). In the PQ
+ HW group, some inflammatory cell infiltration was
observed; however, compared with the PQ group, the
infiltration was lower, the sites of origin were smaller
(Figure 3B). In the PQ group, there are slight
emphysematous changes (Figure 3C).
Partial fibrosis had begun developing in the PQ group
(Figure 3D, D-1). Fibrosis was either not present or only
mild in the PQ + HW group (Figure 3E).
Discussion
Treatments such as gastrointestinal lavage, the
administration of activated charcoal, hemodialysis, and
direct hemoperfusion are often performed in cases of PQ
poisoning. However, there is no evidence that these
methods can ameliorate pulmonary injury.16 Pulmonary
injuries are substantially due to pulmonary fibrosis.
Studies have pointed out that PQ-induced oxidative stress
is involved in pulmonary fibrosis, therefore, a specific
antidote to inhibit this mechanism is currently being
investigated. Superoxide dismutase (SOD), liposomal
encapsulated SOD, the anti-inflammatory actions of
cyclophosphamide and glucocorticoids, the antioxidative
13
Sato, et al.
postcardiac arrest brain injury.34 A randomized doubleblind placebo-controlled trial of Parkinson's disease has
resulted in amelioration, based on the Total Unified
Parkinson's Disease Rating Scale.12 Furthermore, clinical
studies of ischemic-reperfusion injury in patients with
acute myocardial infarction and patients with
postresuscitation complications after cardiopulmonary
resuscitation outside of the hospital are currently being
conducted. The effects of HW on acute PQ poisoning
have been shown in studies using rats.35 In the study,
both biochemical findings (amount of pleural effusion,
number of cells, amount of protein and LDH in BALF
[bronchoalveolar lavage fluid], and MDA levels in
pulmonary tissue) and histologic findings indicate the
effect 72 hours after PQ administration.
To study the effects of HW on mice, we used a
flexiVent to examine the pulmonary functions and
pulmonary histologic findings 3 weeks after the
administration of PQ when pulmonary fibrosis occurs.
Several studies have used flexiVent in assessing various
models for pulmonary fibrosis. When pulmonary fibrosis
is induced, a decrease in C and increases in R, E, Rn, G,
H, and eta were observed.36-38
The present study used Snap Shot to show that E was
significantly lower in the PQ + HW group compared
with that in the PQ group. Although no statistically
significant difference were observed, higher C values
were observed in the PQ + HW group compared with
those in the PQ group. This finding suggests that the
lungs of the PQ + HW group had less resistance than
those of the PQ group, thus facilitating in their inflation.
However, this observation also reflects the condition of
the respiratory system, including the lungs, trachea, and
parenchymal organs; therefore, requires details that
distinguish the central airway, peripheral tissue, and
respiratory functions.
Rn is measured in the central airway by FOT. And H,
G, and eta can be calculated through the peripheral
airways. When fibrosis of the peripheral airways
progresses, H, G, and eta increase. But there are many
points that are not elucidated regarding eta. It is thought
that eta is influenced by various pulmonary mechanical
conditions because eta is the ratio G/H or the area in the
pressure volume loop. Emphysematous changing or
expansion of the peripheral airway with fibrosis has been
observed in the chronic phase of PQ-induced lung injury.
In this study, there were slight emphysematous changes
in the histopathology in the PQ group (Figure 3C).39
Because of this, it will, perhaps, be observed that G and
H do not change, but eta increased in FOT as a result of
combined mechanical change, which is like combined
pulmonary fibrosis and emphysema. Eta should not be
evaluated alone, but the assessment of both G and H are
more important.
In terms of the parameters that did not show a
significant difference, we speculate that the low
concentration of PQ was responsible for these findings.
Approximately 0.5 mg/ml of PQ was administered;
however, because 33% of the mice died within 5 days, a
0.25 mg/ml PQ concentration was utilized in the present
study. This may have been the reason that the level of
lung damage was relatively mild. The selection of amount
and PQ administration method to induce pulmonary
fibrosis was extremely difficult.
Histologic findings of PQ-induced pulmonary injury
by H&E staining showed interstitial edema, pneumocyte
hemorrhage, and interstitial inflammation in early stages
and an increase in fibroblasts and hypertrophy of alveolar
wall cells after 2 or 3 weeks. Furthermore, Azan staining
revealed an accumulation of collagen, which is a
characteristic of pulmonary fibrosis.40
In the present study, H&E and Azan staining revealed
less inflammatory cell infiltration and fibrosis in the PQ
+ HW group compared with that in the PQ group. Because
the histologic findings could not be quantified, the effects
cannot be fully affirmed; however, the findings suggest
that HW was effective for the treatment of PQ-induced
pulmonary injury. These findings suggest that HW lessen
PQ-induced pulmonary injury. Because HW is safe and
imparts no side effects, its intake is easier than that of
other medicines. Further studies are required to determine
whether HW can be established as a treatment regimen
for PQ poisoning in humans.
Acknowledgements
We thank the departments of pharmacology and forensic
medicine at Kitasato University School of Medicine for
the use of their equipment and providing technical advice
for our experiments. We also thank Tetsuo Mikami,
MD, PhD, of the Department of Pathology, Toho
University School of Medicine, for advice concerning
our histological assessments. We especially thank Shigeo
Ohta, MD, PhD, of the Department of Biochemistry and
Cell Biology of Nippon Medical School, Graduate School
of Medicine, Institute of Development and Aging
Sciences for valuable technical advice regarding our
experiments.
14
Hydrogen water for paraquat-induced pulmonary fibrosis in mice
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